PROJECT SUMMMARY Telomeres protect the ends of chromosomes, but shorten naturally with age, leading to their dysfunction. Substantial evidence indicates that telomere dysfunction contributes to major age- related diseases including cancer. Using a mouse model deficient in the telomere length maintenance enzyme telomerase (mTerc-/- mutants), we have discovered a novel positive feedback loop in the intestinal stem cell niche involving telomere capping and the activity of the extracellular Wnt signaling pathway: capping supports Wnt pathway activity, and vice-versa. Thus, telomere shortening in the mouse model leads to inhibition of the Wnt pathway, which leads to further telomere uncapping, ultimately resulting in stem cell dysfunction. Pharmacologic stimulation of Wnt pathway signaling disrupts this vicious cycle, restoring telomere capping and intestinal stem cell function. Therefore pharmacologic or cell-based activation of the Wnt pathway might be of benefit in human disorders caused by telomere dysfunction, but differences in mouse and human telomere biology raise some questions about the translation of our findings from mice to humans. We therefore propose to generate a novel in vitro cell culture model to address the consequences of telomere dysfunction in a human tissue, including potential amelioration by Wnt pathway activation. The model is based on mutations causing dyskeratosis congenita (DC), a genetic disease characterized by decreased telomerase activity and, like mTerc-/- mice, development of intestinal and other pathologies. We propose to utilize cell reprogramming, genetic editing, and directed differentiation technologies to model the intestinal pathology found in DC patients, as well as testing for its rescue by Wnt pathway activators and telomerase activators. This model will help us to understand the pathogenesis and potential treatment of not only DC, but also - more broadly - common diseases in which telomere dysfunction plays a contributory role. Furthermore, the model will provide proof-of- principal findings for future exploration of telomere-related defects in other tissues that can be modeled in culture (e.g. hematopoietic tissues), for screens of additional small molecules that may rescue telomere defects, and for testing the capacity of mutant cells to be genetically repaired and enhance tissue function when transplanted into mouse models.